Molecular Microbiology - 2011 - Hiron - Bacitracin and Nisin Resistance in Staphylococcus Aureus A Novel Pathway Involving

Molecular Microbiology (2011) 81(3), 602–622 䊏 doi:10.1111/j.1365-2958.2011.07735.
x
First published online 4 July 2011
Bacitracin and nisin resistance in Staphylococcus aureus:

a novel pathway involving the BraS/BraR two-component
system (SA2417/SA2418) and both the BraD/BraE and
VraD/VraE ABC transporters mmi_7735 602..622
Aurélia Hiron,1,2 Mélanie Falord,1,2 Jaione Valle,3 cation module and is sufficient to confer bacitracin
Michel Débarbouillé1,2 and Tarek Msadek1,2* and nisin resistance when produced on its own. We
1
Institut Pasteur, Biology of Gram-Positive Pathogens, show that these processes require functional BraD
Department of Microbiology, 25 rue du Docteur Roux, and VraD nucleotide-binding domain proteins, and
75724 Paris Cedex 15, France. that the large extracellular loop of VraE confers its
2
CNRS, URA 2172, F-75015 Paris, France. specificity in bacitracin resistance. This is the first
3
Instituto de Agrobiotecnología, Universidad Pública de example of a TCS associated with two ABC transport-
Navarra-CSIC, Pamplona, 31006, Spain. ers playing separate roles in signal transduction and
antibiotic resistance.
Summary
Introduction
Two-component systems (TCSs) are key regulatory
pathways allowing bacteria to adapt their genetic Staphylococcus aureus is a major Gram-positive human
expression to environmental changes. Bacitracin, a pathogen responsible for a broad spectrum of infections,
cyclic dodecylpeptide antibiotic, binds to undeca- ranging from superficial skin abscesses to more serious
prenyl pyrophosphate, the lipid carrier for cell wall diseases such as pneumonia, meningitis, endocarditis,
precursors, effectively inhibiting peptidoglycan septicaemia and toxic shock syndrome (Lowy, 1998). The
biosynthesis. We have identified a novel and previ- heterogeneity of these diseases and the unique ability of
ously uncharacterized TCS in the major human S. aureus to adapt to a great variety of environments
pathogen Staphylococcus aureus that we show to have made it one of the main causes of hospital- and
be essential for bacitracin and nisin resistance: the community-acquired infections. Treatment of staphylo-
BraS/BraR system (Bacitracin resistance associated; coccal infections is becoming increasingly difficult due to
SA2417/SA2418). The braRS genes are located the worldwide emergence of multiple antibiotic resistance
immediately upstream from genes encoding an ABC determinants (Lowy, 2003), emphasizing the need to
transporter, accordingly designated BraDE. We have further our understanding of the molecular mechanisms
shown that the BraSR/BraDE module is a key bacitra- involved in antimicrobial resistance.
cin and nisin resistance determinant in S. aureus. In Bacitracin, a branched cyclic dodecylpeptide antibiotic
the presence of low antibiotic concentrations, BraSR synthesized by Bacillus licheniformis and some strains
activate transcription of two operons encoding ABC of Bacillus subtilis (Johnson et al., 1945; Azevedo et al.,
transporters: braDE and vraDE. We identified a highly 1993; Ishihara et al., 2002), is active against Gram-
conserved imperfect palindromic sequence upstream positive bacteria and used to prevent and treat skin
from the braDE and vraDE promoter sequences, and ophthalmic infections. Bacitracin binds very tightly
essential for their transcriptional activation by BraSR, to undecaprenyl pyrophosphate (UPP) (Stone and
suggesting it is the likely BraR binding site. We dem- Strominger, 1971), the lipid carrier responsible for trans-
onstrated that the two ABC transporters play distinct location of cell envelope precursors from the cytosol to the
and original roles in antibiotic resistance: BraDE is extracellular surface of the cytoplasmic membrane, effec-
involved in bacitracin sensing and signalling through tively inhibiting peptidoglycan biosynthesis and leading to
BraSR, whereas VraDE acts specifically as a detoxifi- cell lysis.
Gram-positive bacteria have developed several mecha-
Accepted 9 June, 2011. *For correspondence. E-mail tmsadek@ nisms of bacitracin resistance (Cain et al., 1993; Chalker
pasteur.fr; Tel. (+33) 1 45 68 88 09; Fax (+33) 1 45 68 89 38. et al., 2000; Bernard et al., 2005). Among these, the
© 2011 Blackwell Publishing Ltd
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Multi-component bacitracin resistance pathway in Staphylococcus aureus 603
B. subtilis BceSR two-component system (TCS)/BceAB whose genes are located upstream from those of a puta-
ABC transporter is the most efficient and well studied, tive ABC transporter (SA2416/SA2415). While the exist-
constituting the prototype for TCS/ABC transporter ence of an inducible bacitracin resistance mechanism in
modules, where the genes are genetically linked (Ohki S. aureus had been suggested as early as 1949 (Stone,
et al., 2003). TCSs are one of the principal means through 1949), no specific orthologue of the BceSR/BceAB
which bacteria sense, respond and adapt to changes in module has been described in S. aureus. However,
their environment, and are typically composed of a mem- mutants lacking the VraDE ABC transporter, closely
brane histidine kinase (HK), acting as a signal sensor/ related to BceAB but not genetically linked to TCS-
transducer through phosphorylation of its cognate encoding genes, were shown to have increased bacitracin
response regulator (RR), which acts as a transcription sensitivity (Sass et al., 2008; Pietiäinen et al., 2009).
activator or repressor. The BceS HK belongs to the In this study we identified a novel TCS encoded by the
so-called ’intramembrane-sensing kinase’ (IMSK) sub- SA2417/SA2418 genes as a key regulatory element
family, recently defined as conserved in low G+C % Gram- allowing bacitracin and nisin resistance in S. aureus,
positive bacteria and characterized by a very short which we have named BraSR. While this manuscript was
amino-terminal sensing domain, composed of two trans- under review, independent reports have since referred to
membrane helices separated by a small loop of only a few this system as BceSR or NsaSR (Blake et al., 2011;
amino acids, which is thought to be buried in the cytoplas- Yoshida et al., 2011), but we believe the BraSR nomen-
mic membrane (Mascher, 2006). These structural fea- clature to be more appropriate (see Discussion). In the
tures suggest a kinase sensing process occurring at or presence of bacitracin or nisin, we show that BraSR
from within the membrane interface, and members of this activate transcription of the braDE and vraDE operons,
family appear to be involved in responding to cell enve- encoding two ABC transporters, which play distinct and
lope stress, very often exerted by cell wall active antibiot- original roles in bacitracin and nisin resistance. Indeed,
ics (Mascher, 2006; Mascher et al., 2006; Jordan et al., BraDE is only involved in antibiotic sensing and signalling
2008). BceSR control the synthesis of the ABC trans- through BraSR, whereas VraDE acts specifically as a
porter composed of BceA, the nucleotide-binding domain detoxification module and is sufficient to confer bacitracin
(NBD) protein, and BceB, the membrane-spanning and nisin resistance when expressed on its own. This is
domain (MSD) protein or permease, with has 10 trans- the first example of a TCS associated with two ABC trans-
membrane domains and a long extracytoplasmic loop porters involved in separate functions in signal transduc-
(202 aa) (Mascher et al., 2003; Ohki et al., 2003; Rietköt- tion and antibiotic resistance.
ter et al., 2008). In B. subtilis this transporter was shown
to be essential for bacitracin sensing by the BceSR TCS,
highlighting a new ABC transporter-dependent mecha- Results
nism for TCS-mediated signal transduction (Bernard
Identification of a new TCS/ABC transporter module
et al., 2007).
involved in bacitracin and nisin resistance in S. aureus
Most S. aureus strains are endowed with 16 sets of
genes encoding TCSs, with an additional one present in Of the 16 TCSs present in S. aureus, only two have
the staphylococcal cassette chromosome mec of MRSA, IMSKs with the characteristic features of the Bce-like
linked to induction of methicillin resistance (Kuroda et al., family: GraSR, involved in cationic antimicrobial peptide
2001). This sophisticated arsenal of environmental moni- resistance (Herbert et al., 2007) and the uncharacterized
toring proteins could, in part, explain the highly adaptive SA2417/SA2418 system. Indeed, the genes encoding
nature of S. aureus. Although many of these systems these two TCSs are adjacent to those encoding the
remain to be characterized, several have been shown to VraFG and SA2416/SA2415 ABC transporters, respec-
play a role in virulence (AgrCA, SaeSR) (Giraudo et al., tively, and both the GraS and SA2417 kinases have
1999; Novick, 2003), antibiotic resistance (VraSR, extremely short extracellular loops (seven and three
GraSR) (Gardete et al., 2006; Herbert et al., 2007; Meehl amino acids in length respectively).
et al., 2007) or cell wall metabolism (WalKR) (Martin et al., We have constructed a complete collection of S. aureus
1999; Dubrac and Msadek, 2004; Dubrac et al., 2007; mutants inactivated for each of the TCS-encoding gene
Dubrac et al., 2008; Dubrac and Msadek, 2008). Only two pairs (J. Valle, A. Hiron and M. Débarbouillé, unpubl.
of the S. aureus TCSs with IMSKs form genetically linked results) in the HG001 reference strain (Herbert et al.,
TCS/ABC transporter modules sharing some characteris- 2010). Mutants lacking the VraFG, VraDE and SA2416/
tics with the BceSR/BceAB system: GraSR/VraFG, SA2415 ABC transporters were also constructed. Indeed,
playing an essential role in antimicrobial peptide resis- both the VraDE ABC transporter and the VraSR TCS,
tance (Herbert et al., 2007; Li et al., 2007; Meehl et al., whose kinase belongs to the LiaS-like IMSK family
2007), and the uncharacterized SA2417/SA2418 TCS (Mascher, 2006), have been reported to be involved in
© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 81, 602–622

604 A. Hiron et al. 䊏
Table 1. Role of S. aureus two-component systems and ABC trans- bacitracin, indicating that SA2419 is co-transcribed as an
porters in bacitracin and nisin resistance. operon with the braRS genes and that the braDE genes
MIC MIC
are co-transcribed as a separate operon (Fig. 1A and data
bacitracin nisin not shown). A DSA2419 mutant strain was constructed
Strain Relevant genotype (mg ml-1) (mg ml-1) and we showed that this gene does not play a role in
HG001 NCTC 8325 rsbU + 48 >128 bacitracin resistance (data not shown). Primer extension
ST1163 DvraSR 24 >128 experiments were performed to identify the transcription
ST1036 DgraRS 48 4 start sites of the braDE and vraDE operons (Fig. 1B),
ST1179 DbraRS 6 4
ST1090 DvraDE 6 4 which were found to be preceded by appropriately spaced
ST1068 DvraFG 48 4 -10 and -35 regions sharing similarities with the consen-
ST1097 DbraDE 6 4 sus sequence of promoters recognized by the vegeta-
tive form of RNA polymerase, EsA, particularly the -10
sequences, whereas the -35 regions were less conserved
bacitracin resistance in S. aureus (Pietiäinen et al., 2009). (Fig. 1C).
The sensitivity of each mutant strain to bacitracin was
tested (Table 1), allowing us to identify SA2417/SA2418
Expression of the braDE and vraDE operons is induced
as a novel TCS essential for bacitracin resistance, which
by bacitracin or nisin
we have accordingly renamed BraS/BraR (Bacitracin
resistance associated Sensor/Regulator). As shown in In the BceRS/BceAB module of B. subtilis, the bceAB
Table 1, mutants lacking the SA2416/SA2415 genes, genes encoding the ABC transporter are strongly induced
encoding an ABC transporter, also displayed hypersensi- in the presence of sublethal concentrations of bacitracin
tivity to bacitracin, and were renamed braD and braE (Ohki et al., 2003). Since the braDE, vraDE and vraFG
respectively. As expected, the vraDE mutant and, to a operons are all involved in bacitracin and nisin resistance,
lesser extent, the vraSR mutant were also more sensitive we investigated their expression during growth in the
to bacitracin in our genetic background, whereas neither presence of a wide range of subinhibitory concentrations
the absence of the GraSR TCS, which is the most closely of these two antibiotics. Transcriptional lacZ fusions with
related to the B. subtilis BceS/BceR bacitracin resistance the three operon promoters were constructed using the
TCS, nor the absence of the associated VraFG ABC pSA14 vector and introduced into S. aureus strain HG001
transporter affected bacitracin resistance (Table 1). (see Experimental procedures). b-Galactosidase acti-
To determine the substrate specificity of the previously vities were measured during mid-exponential growth
uncharacterized BraSR TCS, sensitivity of the DbraRS (OD600 = 2) with increasing sublethal bacitracin and nisin
mutant strain to a wide spectrum of antimicrobial agents concentrations (ranging from 0.06 to 2 mg ml-1), and are
affecting different cellular processes was tested. No shown as fold induction with respect to the reference
difference in vancomycin, fosfomycin, oxacillin, colistin, value of braDE′–lacZ or vraDE′–lacZ expression in bac-
capreomycin, viomycin or daptomycin sensitivity was teria grown in TSB without antibiotic as an inducer
observed between the wild-type and mutant strain (data (approximately 15 Miller units per mg protein for braDE
not shown) but the DbraRS mutant exhibited highly and 5 for vraDE). We showed that expression of the
increased sensitivity to nisin (Table 1). We also deter- braDE genes is significantly induced in the presence of
mined the nisin minimal inhibitory concentration (MIC) low bacitracin concentrations (ninefold at 0.5 mg ml-1) but
values for mutants lacking the other TCS/ABC modules not by nisin (Fig. 2A). Expression of vraDE was induced
and showed that both the DvraDE and DbraDE strains are by both bacitracin and nisin, even at low concentrations
highly sensitive to nisin (Table 1), as well as the DgraRS (Fig. 2B), but the level of induction was much stronger
and DvraFG mutants, as previously described (Li et al., with bacitracin than with nisin (500-fold and 7-fold at
2007). 0.5 mg ml-1 respectively). Expression from the vraFG pro-
moter on the other hand was not induced by either of the
two antibiotics under these conditions (data not shown).
Genetic structure and transcriptional organization
of the bra and vraDE loci
The BraSR TCS controls inducible expression of
Close examination of the braRS locus indicates that the
the braDE and vraDE operons but does not regulate
two genes are located 25 bp downstream from a gene
its own synthesis
annotated as encoding a 66-amino-acid hypothetical
protein (SA2419) and 107 bp upstream from the braDE As shown above, the BraSR, VraSR and GraSR TCSs are
genes (Fig. 1A). We performed RT-PCR analysis on total each involved to various extents in bacitracin and nisin
RNA extracted from cells grown in TSB with 0.5 mg ml-1 resistance. We therefore wished to determine which of

Fig. 1. Genetic structure and primer extension analysis of the braRS, braDE and vraDE loci.
A. Genetic organization of the braRS and braDE operons. Genes are indicated by grey arrows, operon transcripts by double-headed
horizontal arrows and the braDE operon transcription initiation site by a horizontal arrow.
B. Primer extension analysis of braDE and vraDE mRNAs. Total RNA isolated from strain HG001 grown in TSB with 0.5 mg ml-1 bacitracin was
used as a template for primer extension experiments using braD- and vraD-specific radiolabelled oligonucleotides. The corresponding Sanger
dideoxy chain termination sequencing reactions were carried out on a PCR fragment corresponding to the respective operon upstream regions
(lanes C, T, A, G).
C. Nucleotide sequences of the braDE and vraDE operon promoter regions. The -35 and -10 promoter sequences are boxed, the
transcription initiation start sites are labelled +1 and the ATG translation initiation codons are indicated in bold.
these systems might be involved in controlling braDE and vraDE expression in the presence of nisin (Fig. 3B).
vraDE expression in response to these antibiotics. Plas- These data clearly demonstrate that induction of the
mids carrying braDE′–lacZ and vraDE′–lacZ transcrip- vraDE and braDE operons in response to bacitracin or
tional fusions were introduced into the DvraSR, DgraRS nisin is mediated by the BraSR TCS but is not controlled
and DbraRS mutant strains, and b-galactosidase assays by VraSR or GraSR. We also verified that expression of
were performed during growth in TSB with or without vraFG was not affected in the DbraRS mutant in the
0.5 mg ml-1 bacitracin or nisin. Expression of braDE and presence or absence of bacitracin (data not shown).
vraDE was no longer induced by bacitracin in the DbraRS The DbraRS mutant was complemented by expressing
mutant while no significant difference with the parental the braRS genes from the pMK4-Pprot multicopy plasmid
strain was observed in the DvraSR and DgraRS mutant (Archambaud et al., 2005), fully restoring resistance to
strains (Fig. 3A and B). Similar results were obtained for bacitracin and nisin (Table 2), as well as bacitracin-

Fig. 2. Induction of vraDE and braDE expression as a function of bacitracin and nisin concentrations. Antibiotic concentrations (mg ml-1) are
shown on the x-axis and plotted against fold induction values (y-axis). The reference expression value is that of bacteria grown in TSB without
antibiotic. Black bars represent induction in the presence of bacitracin and grey bars in the presence of nisin. b-Galactosidase assays were
performed as described in Experimental procedures. Results are presented as the average of three independent assays. The strains used for
promoter activity measurements are (A) ST1245: pSA14-PbraDE–lacZ and (B) ST1208: pSA14-PvraDE–lacZ.
Fig. 3. Inducible expression of the braDE and vraDE operons is controlled by the BraSR TCS. b-Galactosidase assays (A) and (B) and
qRT-PCRs (C) were performed as described in Experimental procedures. Cells were grown until mid-exponential phase (OD600 = 2).
b-Galactosidase activities and qRT-PCR products on the y-axis are represented as fold induction against the reference value, i.e. that of
bacteria grown in TSB in the absence of bacitracin or nisin. Black and grey bars represent expression levels in the presence of 0.5 mg ml-1
bacitracin or 0.5 mg ml-1 nisin respectively.
A. Effect of braRS, vraSR and graRS deletions on PbraDE expression. The strains used are (from left to right): ST1245 (pSA14-PbraDE–lacZ),
ST1087 (DbraRS pSA14-PbraDE–lacZ), ST1092 (DvraSR pSA14-PbraDE–lacZ), ST1091 (DgraRS pSA14-PbraDE–lacZ). Results are presented as
the average of three independent assays.
B. Effect of braRS, vraSR and graRS deletions on PvraDE expression. The strains used are (from left to right): ST1208 (pSA14-PvraDE–lacZ),
ST1088 (DbraRS pSA14-PvraDE–lacZ), ST1092 (DvraSR pSA14-PvraDE–lacZ), ST1089 (DgraRS pSA14-PvraDE–lacZ). Results are presented as the
average of three independent assays.
C. Relative levels of vraDE transcripts were measured by qRT-PCRs. Expression levels were normalized using 16S rRNA as an internal
standard and are indicated as n-fold change, expressed as the means and standard deviations of quadruplicate experiments. The strains used
are (from left to right): HG001 (reference strain), ST1179 (DbraRS) and ST1184 (DbraRS pMK4-Pprot braRS).

Table 2. Effects of vraDE and braDE complementation on bacitracin and nisin resistance.
Strain Relevant genotype MIC bacitracin (mg ml-1) MIC nisin (mg ml-1)
HG001 NCTC 8325 rsbU+ 48 > 128

ST1216 D braDE pMK4-Pprot 6 4
ST1187 D braDE pMK4-Pprot braDE 48 > 128
ST1188 D braDE pMK4-Pprot vraDE 16 > 128
ST1232 D braDE pMK4-Pprot braR 32 > 128
ST1217 D vraDE pMK4-Pprot 6 4
ST1192 D vraDE pMK4-Pprot braDE 6 4
ST1191 D vraDE pMK4-Pprot vraDE 16 > 128
ST1233 D vraDE pMK4-Pprot braR 6 4
ST1238 D braRS pMK4-Pprot 6 4
ST1185 D braRS pMK4-Pprot braDE 6 4
ST1186 D braRS pMK4-Pprot vraDE 16 > 128
ST1184 D braRS pMK4-Pprot braRS 48 > 128
ST1121 D braRS pMK4-Pprot braR 32 > 128
ST1239 D vraDE D braDE pMK4-Pprot 6 4
ST1240 D vraDE D braDE pMK4-Pprot braDE 6 4
ST1218 D vraDE D braDE pMK4-Pprot vraDE 16 > 128
dependent induction of the vraDE genes as determined directed mutagenesis through PCR in one of the
by quantitative real-time PCR (qRT-PCR) (Fig. 3C). inverted repeats, effectively destroying the palindromic
As many TCSs are known to positively autoregulate operator sequence and leading to the complete loss of
their own synthesis, a transcriptional lacZ fusion with the induction of expression from the braDE and vraDE pro-
SA2419/braRS operon promoter region was constructed. moters by bacitracin or nisin (Fig. 4A–C). Together,
The SA2419/braRS′–lacZ fusion was expressed constitu- these results indicate that the conserved inverted repeat
tively in the absence of bacitracin (approximately 400– upstream from the vraDE and braDE promoters is
500 Miller units per mg protein), and this expression did essential for the induction of promoter activity in the
not vary in the presence of bacitracin concentrations presence of bacitracin and nisin, strongly suggesting it is
ranging from 0.0625 to 2 mg ml-1 or in the DbraRS mutant the likely binding site for the BraR RR. Genome scan-
strain ST1179, indicating that BraSR do not control their ning analysis, using the AureoList relational database
own synthesis (data not shown). (http://genolist.pasteur.fr/AureoList/), indicates that no
S. aureus genes other than the braDE and vraDE
operons are preceded by this sequence.
Identification of a conserved palindromic sequence as
the likely BraR binding site
The BraDE ABC transporter is involved in
We have shown that expression of the vraDE and braDE
antibiotic detection
operons is controlled by BraSR in response to antibiot-
ics. An in silico approach was used to identify potential In B. subtilis, the BceAB ABC transporter has been shown
regulatory DNA motifs in the promoter regions of these to be required for its own synthesis (Bernard et al., 2007).
two co-regulated operons. A highly conserved imper- As shown above, two separate ABC transporters, VraDE
fect 14-base-pair palindromic motif (CTTTCAA NN and BraDE, are both involved in S. aureus bacitracin and
T/CTGTAAG) was identified, located 66 and 64 bp nisin resistance. In order to determine their respective
upstream from the braDE and vraDE transcription start contributions in signal perception and antibiotic resis-
sites respectively (Fig. 4A). To test whether this con- tance, braDE′–lacZ and vraDE′–lacZ expression was
served sequence is necessary for expression of the two measured in DvraDE and DbraDE mutant strains. As
operons in response to bacitracin and/or nisin, a series shown in Fig. 5A and B, the BraDE ABC transporter is
of truncated promoter fragments were generated and essential for bacitracin and nisin-dependent transcription
used to construct transcriptional lacZ fusions using from both the braDE and vraDE promoters, whereas the
plasmid pSA14 (Fig. 4B). Removal of the braDE and VraDE ABC transporter was not required. To confirm
vraDE upstream sequences up to the inverted repeat these results, expression of the braDE and vraDE genes
had no effect on promoter activity; however, removal of was uncoupled from BraSR-dependent regulation using
half or all of the inverted repeat completely abolished the pMK4-Pprot plasmid, and used to complement the
bacitracin- and nisin-induced expression (Fig. 4B and DbraDE and DvraDE mutant strains respectively. Expres-
C). Three point mutations were introduced by site- sion of vraDE was measured by qRT-PCR in the wild-type

Fig. 4. A highly conserved imperfect

palindromic sequence within the braDE and
vraDE promoters is required for
bacitracin-dependent promoter activation.
A. Alignment of the braDE and vraDE
promoter region nucleotide sequences. The
highly conserved palindromic sequence is
shaded and boxed and indicated by inverted
arrows. Point mutations introduced in the
braDE ′ and vraDE ′–lacZ fusions are indicated
below the alignment by stars. Nucleotide
positions are given with respect to the
transcription initiation sites.
B and C. b-Galactosidase assays of braDE
(B) and vraDE (C) promoter transcriptional
fusions. The braDE and vraDE promoter
regions were deleted up to the inverted repeat
(DB), half way through the inverted repeat
(DC), just after the potential BraR binding site
(DD) or carried 3 point mutations destroying
the inverted repeat (DE). Positions are
numbered with respect to the transcription
initiation site. S. aureus strains carrying the
indicated transcriptional fusions were grown in
triplicate in TSB with 0.5 mg ml-1 bacitracin
(black bars) or 0.5 mg ml-1 nisin (grey bars).
b-Galactosidase activities are given as the
average of three independent assays and
represented as fold induction with respect to
the reference value, i.e. braDE′–lacZ or
vraDE′–lacZ activity of bacteria grown in TSB
without antibiotic as an inducer.
strain HG001, the DbraDE mutant strain (ST1097) and the this partial complementation of bacitracin resistance in the
DbraDE complemented strain (ST1187), grown in the DvraDE mutant, vraDE and braDE mRNA levels were
presence of bacitracin (0.5 mg ml-1). As shown in Fig. 5C, compared by qRT-PCR in the wild-type and comple-
the BraDE ABC transporter is essential for induction of mented strains. In comparison with the wild-type strain in
vraDE expression by bacitracin. Taken together, these the presence of bacitracin, expression levels of braDE
results indicate that only the BraDE ABC transporter is from the pMK4-Pprot plasmid were threefold higher,
essential for bacitracin and nisin detection by BraSR. whereas plasmid-based expression of vraDE was 3.5-fold
lower in the DvraDE complemented strain. This result is
consistent with our observation that expression of the
The VraDE ABC transporter acts as a
vraDE genes is much more highly induced in the pres-
resistance module
ence of 0.5 mg ml-1 bacitracin than that of braDE (approxi-
We then addressed the question of the role of the BraDE mately 500-fold and 7-fold respectively) (Fig. 1).
and VraDE ABC transporters in antibiotic detoxification. The pMK4-PprotbraDE and pMK4-PprotvraDE plas-
Complementation by the pMK4-Pprot braDE plasmid fully mids were introduced into different mutant strains as
restored bacitracin and nisin resistance to the DbraDE shown in Table 2 and sensitivity to bacitracin and nisin
mutant and the pMK4-Pprot vraDE plasmid restored sig- was compared. Interestingly, constitutive expression of
nificant levels of bacitracin resistance and full resistance vraDE on its own bypasses the requirement for BraSR
to nisin to the DvraDE mutant strain (Table 2). To explain and BraDE (Table 2) restoring bacitracin and nisin

Fig. 5. BraDE is required for bacitracin-dependent induction of braDE and vraDE expression. b-Galactosidase assays (A and B) and
qRT-PCRs (C) were performed as described in Experimental procedures. Cells were grown until mid-exponential phase (OD600 = 2).
b-Galactosidase activities and qRT-PCR products are represented as fold induction against the reference value, i.e. bacteria grown in TSB
without antibiotic as an inducer. Black and grey bars represent expression levels in the presence of 0.5 mg ml-1 bacitracin or 0.5 mg ml-1 nisin
respectively.
A. Effect of braRS, braDE and vraDE deletions on PbraDE expression. The strains used are (from left to right): ST1245 (pSA14-PbraDE–lacZ),
ST1087 (DbraRS pSA14-PbraDE–lacZ), ST1189 (DbraDE pSA14-PbraDE–lacZ), ST1195 (DvraDE pSA14-PbraDE–lacZ).
B. Effect of braRS, braDE and vraDE deletions on PvraDE expression. The strains used are (from left to right): ST1208 (pSA14-PvraDE–lacZ),
ST1088 (DbraRS pSA14-PvraDE–lacZ), ST1190 (DbraDE pSA14-PvraDE–lacZ), ST1230 (DbraD pSA14-PvraDE–lacZ), ST1196 (DvraDE
pSA14-PvraDE–lacZ).
C. Relative levels of vraDE transcripts were measured by qRT-PCR. Expression levels were normalized using 16S rRNA as an internal
standard and are indicated as n-fold change with respect to those of bacteria grown in TSB in the absence of bacitracin, expressed as the
means and standard deviations of quadruplicate experiments. The strains used are (from left to right): HG001, ST1097 (DbraDE) and ST1187
(DbraDE pMK4-Pprot braDE).
resistance. In the reverse experiment, expression of last point, the braR gene was expressed alone using the
braDE could not complement the DbraRS and DvraDE pMK4-Pprot plasmid. Overexpression of RR genes is well
mutant stains (Table 2). known to bypass the requirement for the cognate HK,
These results suggest that the VraDE ABC transporter leading to constitutive activation due to other phosphate
plays a direct role in the bacitracin detoxification process donors such as acetyl phosphate or aspecific kinase
whereas BraDE is only required for bacitracin sensing activity within the cell (Kobayashi et al., 2001). As shown
and/or signal transduction through BraSR. To confirm this in Table 2, the pMK4-PprotbraR plasmid restores signifi-

Table 3. BraD- and VraD-dependent bacitracin resistance requires a functional ATP-binding domain.
Strain Relevant genotype MIC bacitracin (mg ml-1)
ST1210 DbraD pMK4-Pprot 6

ST1197 DbraD pMK4-Pprot braD 48
ST1206 DbraD pMK4-Pprot braD*WB 6
ST1231 DvraD pMK4-Pprot 6
ST1198 DvraD pMK4-Pprot vraD 16
ST1205 DvraD pMK4-Pprot vraD*WB 6
cant levels of bacitracin resistance to both the DbraRS Taken together, these results strongly suggest that both
and DbraDE mutant strains but was not able to comple- the BraDE and VraDE ABC transporters require ATP
ment the DvraDE mutant (Table 2). These results confirm hydrolysis to exert their respective functions in antibiotic
that, contrary to VraDE, the BraDE ABC transporter only sensing and resistance.
appears to be required for sensing and BraSR-dependent
signal transduction, leading to phosphorylation of the
The extracellular loop of the VraE permease confers its
BraR RR and antibiotic resistance.
specificity for bacitracin resistance
As shown above, VraDE plays a central role in bacitra-

Bacitracin sensing and resistance requires functional
cin resistance. The predicted membrane topology and
BraD and VraD ABC transporter NBD proteins
primary structure of this ABC transporter are very similar
As described above, the BraDE and VraDE ABC trans- to those of VraFG (62% amino acid sequence identity
porters play specific roles in bacitracin detection and between VraD and VraF and 39% between VraE and
resistance respectively. In order to determine whether VraG), whereas the BraDE ABC transporter is not closely
these processes are energy-dependent, the braD or vraD related (Fig. 6). The VraFG system is involved in resis-
genes, encoding the ABC transporter NBD proteins were tance to cationic antimicrobial peptides and its synthesis
deleted. Both NBD mutant strains were as sensitive to is controlled by the GraSR TCS (Li et al., 2007; Meehl
bacitracin as the DbraDE and DvraDE mutant strains et al., 2007). We have shown that neither GraSR nor
(Table 3). As shown in Fig. 5, inducible expression of VraFG is involved in bacitracin resistance (Table 1) and
vraDE and braDE by bacitracin and nisin was abolished in also that vraFG expression is not controlled by BraSR and
the DbraD mutant strain (Fig. 5B). Since the braDE and is not induced in the presence of bacitracin (data not
vraDE genes are co-transcribed as an operon, removing shown). As shown in Fig. 6B, sequence alignment of the
the first gene might have a polar effect on expression of VraE and VraF permeases indicated that the most dissimi-
the downstream permease gene. To rule out that possi- lar sequence between the two proteins corresponds to a
bility, we complemented the DbraD and DvraD mutants large extracytoplasmic loop of respectively 193 and 198
using the pMK4-Pprot plasmid, fully restoring bacitracin amino acids in length, located between transmembrane
resistance (Table 3). We also note that both ATPases regions 7 and 8 (Fig. 6A). Amino acid sequence identity
were highly specific to their cognate permease as no between these two loops is only 20% whereas the rest
cross-complementation between vraD and braD was of the two proteins share 53% identity (Fig. 6B). In
observed (data not shown). B. subtilis, the extracellular loop of BceB is thought to
To investigate whether ATP hydrolysis by BraD or VraD be necessary for bacitracin sensing and resistance
is critical for their role in antibiotic resistance, single-point (Rietkötter et al., 2008; Coumes-Florens et al., 2011).
mutations were introduced by site-directed mutagenesis To investigate the role of the VraDE extracellular loop in
through PCR in the Walker B motif-coding sequences of S. aureus, we took advantage of the close similarities
braD (E168Q) and vraD (E168Q). This mutation has been between VraE and VraG in order to construct a chimeric
described to abolish ATP hydrolysis, but not binding, while ABC transporter by domain-swapping, exchanging the
simultaneously stabilizing the NBD dimer and maintaining extracellular loop of VraG for that of VraE and expressing
the native ATP-bound structure (Moody et al., 2002). The the recombinant gene using the pMK4-Pprot plasmid. The
mutated alleles were expressed using the pMK4-Pprot resulting plasmid, pMK4-PprotvraFG*vraE, was introduced
plasmid (pMK4-PprotbraD*WB and pMK4-PprotvraD*WB into the DvraDE mutant strain and was as efficient in
respectively), and were both unable to complement the restoring bacitracin resistance as the pMK4-PprotvraDE
respective DbraD and DvraD mutants in contrast to the complementing plasmid (Table 4). We verified that the
wild-type alleles (Table 3). pMK4-PprotvraFG plasmid did not confer resistance to

Fig. 6. Topology and sequence comparison between ABC transporter membrane-spanning domain proteins.
A. Membrane topology of VraE, VraG and BraE. The graphical representation of the membrane topology prediction was performed using the
TopPredII program and von Heijne’s algorithm (Claros and von Heijne, 1994). The extracytoplasmic loop between the predicted
membrane-spanning helices 7 and 8 has a respective length of 193, 198 and 231 amino acids for VraE, VraG and BraE.
B. Alignment of the VraE, VraG and BraE amino acid sequences. Sequence alignment was performed using the CLUSTAL program (Higgins
et al., 1992). Identical residues are shaded. The predicted extracellular loops of the VraE and VraG permeases that were exchanged by
domain-swapping to construct the VraFG*VraE chimerical permease are boxed.
bacitracin but was able to fully complement a DvraFG Table 4. The extracellular loop of VraE is essential for bacitracin
mutant strain for resistance to colistin, a cationic antimi- detection.
crobial peptide (Table 4). These results demonstrate that
MIC
the extracellular loop of the VraE permease is the deter- Strain Relevant genotype (mg ml-1)
minant of its specificity in bacitracin resistance. Interest-
Bacitracin
ingly, we also showed that the pMK4-Pprot vraFG*vraE HG001 NCTC 8325 rsbU + 48
plasmid did not restore colistin resistance to a DvraFG ST1068 D vraFG 48
mutant strain (Table 4), confirming the importance of ST1217 D vraDE pMK4-Pprot 6
ST1191 D vraDE pMK4-Pprot vraDE 16
the extracellular loop in the specificity of substrate ST1193 D vraDE pMK4-Pprot vraFG 6
recognition. ST1194 D vraDE pMK4-Pprot vraFG*vraE 16
We also constructed the pMK4-PprotbraDE*vraE ST1234 D vraDE pMK4-Pprot braDE*vraE 6
ST1216 D braDE pMK4-Pprot 6
plasmid, expressing a chimeric system with the BraE ST1235 D braDE pMK4-Pprot braDE*vraE 6
extracellular loop replaced by that of VraE. This plasmid Colistin
could not complement the DbraDE or DvraDE mutant ST1120 HG001 pMK4-Pprot 700
ST1150 D vraFG pMK4-Pprot 100
strains for bacitracin resistance (Table 4). Although we ST1151 D vraFG pMK4-Pprot vraFG 700
cannot exclude that the chimeric BraE*VraE permease ST1177 D vraFG pMK4-Pprot vraFG*vraE 200
may be non-functional because of the greater diversity
vraG*vraE, vraG with the extracellular loop-coding sequence of vraE;
between BraE and VraE, this result could suggest that braE*vraE, braE with the extracellular loop-coding sequence of vraE.
unlike VraDE, the extracellular loop of the permease is not

Fig. 7. Signal transduction network

controlling bacitracin sensing and resistance
in S. aureus. Bacitracin is sensed by BraDE
and the signal is transduced to BraSR
through a mechanism that remains to be
elucidated, but which likely involves
interaction between BraE and BraS. Activation
of the BraSR system leads to
BraS-dependent phosphorylation of BraR and
subsequent transcription activation of the
braDE and vraDE operons. The VraDE
transporter then removes bacitracin. As the
direction of bacitracin transport by VraDE, i.e.
import or export, is not known, it is indicated
by a vertical double-headed arrow, but we
have demonstrated that the extracellular loop
of VraE is a determinant of its specificity and
activity.
the sole determinant involved in bacitracin sensing and cient, sensitive and specific BraSR/BraDE/VraDE multi-
signalling by the BraDE ABC transporter. component system, and the damage-sensing VraSR
system, less effective but responding to general envelope
stress conditions.
Discussion The BraSR and GraSR TCSs share some common
In this study, we identified a novel TCS/ABC transporter features: they both have IMSK family kinases and control
module, BraSR/BraDE, as the main regulatory element the synthesis of closely related ABC transporters (VraDE
controlling bacitracin and nisin resistance in S. aureus. and VraFG) (Fig. 6B), and they are both essential for nisin
We showed that together with VraDE, the BraSR/BraDE resistance. A transcriptome analysis previously suggested
module constitutes an original mechanism for antibiotic that expression of the vraDE operon is controlled by
sensing and resistance. GraSR in response to indolicidin (Li et al., 2007). Indeed,
The BraSR TCS responds to low bacitracin concentra- they observed an induction of approximately ninefold in
tions by inducing expression of the braDE and vraDE vraDE expression upon addition of 0.5 mg ml-1 indolicidin,
genes, encoding two ABC transporters (Fig. 7). The which fell to threefold in a DgraRS mutant, suggesting the
DbraRS, DbraDE and DvraDE mutant strains are all existence of an additional unidentified regulatory system.
highly sensitive to bacitracin (Table 1), suggesting that In order to test whether this residual regulation was due to
the BraSR/BraDE/VraDE system could be functionally BraSR, suggesting a possible co-regulation of vraDE
equivalent to the BceSR/BceAB module of B. subtilis expression by the BraSR and GraSR TCSs, we measured
(Ohki et al., 2003). Among the panel of antibiotics tested vraDE expression in the presence of indolicidin. We saw
in this study, the BraSRDE/VraDE multicomponent no induction of vraDE′–lacZ expression in our genetic
system displayed restricted substrate specificity since it background using indolicidin concentrations ranging from
appears to only be involved in bacitracin and nisin 0.5 to 40 mg ml-1 (data not shown). Moreover, vraDE
resistance. expression did not vary between the WT and DgraRS
In contrast, the VraSR TCS plays a minor role in baci- strains with or without bacitracin, nisin, colistin or indolici-
tracin resistance (Table 1) but the DvraSR mutant is din (Fig. 3B and data not shown). Taken together these
sensitive to a large range of antimicrobial compounds results demonstrate that vraDE expression is clearly not
(Pietiäinen et al., 2009). VraSR is thought to sense cell controlled by the GraSR TCS under our conditions and
wall damage to co-ordinate a general cell envelope stress that no cross-regulation between the Bra and Gra TCSs
response in S. aureus (Belcheva and Golemi-Kotra, exists. In the previous report (Li et al., 2007) no indepen-
2008) and thus could be considered as the equivalent of dent validation of the transcriptome data was reported
the B. subtilis LiaSR system (Mascher et al., 2004). Two and other transcriptome experiments did not identify
distinct pathways contributing to bacitracin resistance can vraDE as a member of the GraSR regulon (Herbert et al.,
therefore be distinguished in S. aureus: the highly effi- 2007).

Contradictory results have also been reported regard- braRS operon promoter drives expression of the intact
ing sensitivity of the S. aureus DvraDE mutant towards braR gene and that of a truncated copy of braS, while a
antibiotics. Indeed, VraDE was first reported as being truncated copy of braR and an intact copy of braS are still
involved in resistance to a large number of antibiotics present downstream from the plasmid insertion, albeit
and/or antimicrobial peptides (daptomycin, bacitracin, separated from the operon promoter (Kolar et al., 2011).
vancomycin, hBD3, LL37, nisin, pep5) (Sass et al., 2008) The transcriptome analysis was performed by comparing
and then as only playing a role in bacitracin resistance, expression levels of genes in this mutant, where expres-
using a similar selection of antibiotics as the previous sion of braS is thought to be reduced, with those in the
study (Pietiäinen et al., 2009). In our genetic background, parental SH1000 strain (Kolar et al., 2011). However, the
we found that, among the panel of tested antibiotics (van- analysis was carried out using cells grown in the absence
comycin, fosfomycin, oxacillin, colistin, capreomycin, of any inducing antibiotic, making it difficult to judge the
viomycin, daptomycin, indolicidin), the VraDE ABC trans- physiological relevance of the results. Indeed, TCSs are
porter is only involved in resistance to bacitracin and nisin, known to be essentially inactive in the absence of the
two cyclic polypeptides that target lipid II precursor or lipid signal detected by the kinase and as we show here,
II (Siewert and Strominger, 1967; Breukink and de Kruijff, expression of genes controlled by BraSR such as the
2006), which could explain that this system has evolved vraDE operon is approximately 500-fold lower in the
as a common defence system against these two com- absence of bacitracin, remaining at background levels. In
pounds in S. aureus. We cannot, however, exclude that fact, in their analysis, Kolar et al. failed to identify vraDE
other lantibiotics besides nisin that target lipid II may also as being controlled by BraSR (Kolar et al., 2011), although
act as inducers for the BraSR TCS. Indeed, both actagar- it is the most highly regulated member of the regulon as
dine and mersacidin were recently shown to be inducers we show here. Furthermore, Kolar et al. saw no effect of
of the B. subtilis BceSR TCS whereas nisin does not, their braS mutation on sensitivity to nisin or bacitracin, in
although the resistance conferred by the system to the contrast to our results and those of Yoshida et al. (Kolar
two former compounds is much lower than to bacitracin et al., 2011; Yoshida et al., 2011) and they report that
(Staron et al., 2011). expression of braRS is induced by nisin, whereas we
Induction of vraDE expression by very high vancomycin show here that expression of braRS is not induced by
concentrations (10- and 5-fold MIC) has also been bacitracin or nisin, and that BraSR do not autoregulate
reported in two transcriptomic studies (Kuroda et al., their own synthesis. Part of the problem may come from
2003; Pietiäinen et al., 2009). We performed the fact that their study was carried out in the S. aureus
b-galactosidase assays with sublethal vancomycin SH1000 strain, derived from strain 8325-4, both of which
concentrations ranging from 0.25 to 1 mg ml-1 are known to contain multiple mutations in comparison
(MIC = 2 mg ml-1) and saw no effect on vraDE expression with the reference NCTC 8325 strain (O’Neill, 2010),
(data not shown). These results indicate that if vraDE whereas we used the HG001 strain, a rsbU-repaired
induction by vancomycin does occur, it is only at very high variant of strain NCTC 8325, which has never been
concentrations and independent of the BraRS/BraDE mutagenized or subjected to UV irradiation (Herbert et al.,
regulatory pathway, which is highly sensitive and 2010).
responds to low concentrations of bacitracin or nisin The appearance of these recent reports raises the
(Fig. 2). issue of nomenclature for this TCS, as it has been referred
The BraSR TCS has attracted a considerable amount of to variously as NsaS/NsaR (Blake et al., 2011) or BceS/
attention recently. Indeed, during the past several months BceR (Yoshida et al., 2011), and in our study as BraS/
while different versions of this manuscript were under BraR. In our view, the Nsa appellation is inappropriate
review, several independent reports appeared in the lit- since we show here that nisin does not induce expression
erature, studying various aspects of the system. In agree- of the BraSR-dependent braDE operon, and only induces
ment with the results reported here, separate studies expression of vraDE approximately sevenfold, whereas
reported BraSR as linked to bacitracin resistance in bacitracin induces both target operons significantly (over
S. aureus strain MW2 (Matsuo et al., 2010; Yoshida et al., 500-fold for vraDE). Additionally, the system was originally
2011), where the system was referred to as BceSR identified by ourselves and Matsuo et al. as linked to
(Yoshida et al., 2011), while missense mutations within bacitracin resistance (Matsuo et al., 2010). The BceSR
braS were described as leading to increased nisin resis- designation is equally inappropriate since BraSR is not
tance in the SH1000 genetic background, and the TCS the orthologue of the B. subtilis BceSR system (see
was designated NsaS/NsaR (Nisin susceptibility associ- above); BceSR-dependent gene expression in B. subtilis
ated) (Blake et al., 2011). is not induced by nisin, nor does the Bce system confer
More recently, a transcriptome analysis was performed resistance to nisin (Staron et al., 2011); and the BraSR
in the SH1000 background using a strain where the system differs significantly in its mechanistics from the

BceSR system in that it uses two distinct ABC transpor- tance process itself. Our results strongly suggest these
ters, one for sensing (BraDE) and one for detoxification two distinct functional roles are both energy-dependent,
(VraDE) as we show here. For all of these reasons, we since the BraD and VraD NBD proteins with intact ATP
believe that the Nsa or Bce nomenclatures would be hydrolysis domains are each essential for bacitracin resis-
misleading and instead propose that the system be des- tance (Table 3). Although BraE and VraE are similar in
ignated BraSR (Bacitracin resistance associated), follow- their predicted membrane topology (Fig. 6A) the proteins
ing the previously established S. aureus nomenclature for share only 33% amino acid identity overall. We demon-
related systems (Gra, Vra). strated that the extracellular loop of VraE is the specificity
The main part of this study was devoted to the charac- determinant for bacitracin recognition, but sequence
terization of the BraSR/BraDE/VraDE system. Following alignment with BraE did not reveal any particularly similar
bacitracin detection, we have shown that BraSR strongly or dissimilar regions (Fig. 6B) in contrast to VraG and
activates transcription of the braDE and vraDE operons VraE, making it difficult to predict a common bacitracin
(Fig. 3). BraR is a member of the OmpR subfamily of RRs, recognition domain between BraE and VraE. The func-
with a typical winged helix–turn–helix domain (Martinez- tional specialization of BraDE and VraDE in the S. aureus
Hackert and Stock, 1997; Mizuno and Tanaka, 1997) bacitracin detoxification process is quite unexpected
extending from amino acid residues 172 to 202. Although since in B. subtilis the BceAB transporter is believed to
OmpR-type RRs are known to bind to short direct repeats carry out both roles, in sensing and transport (Bernard
with a spacing placing them on the same face of the DNA et al., 2007; Rietkötter et al., 2008). Although a role for
helix (Blanco et al., 2002; Dubrac and Msadek, 2004), BceAB in bacitracin sensing has been shown, there is no
several members of this family such as BceR of B. subtilis evidence that it is directly involved in the detoxification
or Streptococcus mutans were in fact shown to bind to process and the bacitracin sensitivity of a B. subtilis
inverted repeat sequences (Ohki et al., 2003; Ouyang bceSR mutant constitutively expressing bceAB was not
et al., 2010). tested. However, we were unable to identify any VraDE
In this study we have identified a conserved imperfect equivalent in B. subtilis, suggesting that the involvement
palindrome (CTTTCAA NN T/CTGTAAG) in the promoter of two distinct ABC transporters with separate functions in
regions of the braDE and vraDE operons and through a bacitracin resistance might be unique to Staphylococcus
genetic approach, combining deletions and point muta- species.
tions within this sequence, shown that it is essential for Our results allowed us to propose a model for antibiotic
bacitracin-dependent induction and activation by BraSR sensing and resistance through this pathway in S. aureus
(Fig. 4). Although the sequence of this operator site is (Fig. 7), where the antibiotic is first detected by the BraDE
strikingly similar to that reported for BceR of S. mutans, ABC transporter, as we have shown that a braDE mutant
strongly suggesting it is indeed the BraR binding site, it strain no longer responds to the presence of extracellular
shares no similarities with the binding site for BceR of B. bacitracin or nisin (Fig. 5). This is fairly unusual since
subtilis (Ohki et al., 2003; Ouyang et al., 2010). The posi- typically in TCSs the signal is sensed by the external loop
tion of the likely BraR binding site, approximately 60 bp of the HK (Hoch and Silhavy, 1995). However the BraS
upstream from the transcription initiation sites of the HK, like BceS of B. subtilis, belongs to the IMSK family.
braDE and vraDE operons (Fig. 4A), suggests that BraR Because of their short extracellular loop (only three amino
is probably a Class I transcription activator, interacting acids in BraS), it has been suggested that these kinases
with the a subunit of RNA polymerase (Ishihama, 1993). detect their stimuli from within or at the membrane inter-
A central finding of this study is the involvement of two face (Mascher, 2006; Mascher et al., 2006). Our results
separate ABC transporters, BraDE and VraDE, playing support the hypothesis that the stimulus is sensed by the
distinct roles in antibiotic resistance: the first being BraDE ABC transporter and then transferred to BraS,
dedicated to signal perception and the second to which in turn activates the BraR RR, as suggested for the
detoxification. Indeed, complementation experiments with BceSR/BceAB module in B. subtilis and S. mutans
constitutively expressed braDE and vraDE genes show (Bernard et al., 2007; Ouyang et al., 2010).
that VraDE is sufficient to confer resistance to bacitracin In this model, several important questions remain to be
and nisin when expressed on its own, whereas BraDE is answered. First, how does the BraDE ABC transporter
not. Conversely, BraDE is required for expression of the sense bacitracin? The membrane topology of the BraE
braDE and vraDE operons, whereas VraDE is not, indi- permease (Figs 6A and 7) is similar to that of BceB in
cating that the VraDE ABC transporter is only involved B. subtilis, characterized by the presence of a large extra-
in the detoxification process. Complementation of the cellular loop, which has been suggested to be essential
DbraDE mutant by overproducing BraR confirmed that for bacitracin detection by BceB (Rietkötter et al., 2008;
this ABC transporter is only required for activation of the Coumes-Florens et al., 2011). It is likely that in BraE, this
RR through phosphorylation and has no role in the resis- extracellular loop is also essential for signal recognition

and could be involved in direct interaction with the bacterial culture grown in TSB to an OD600 = 1 was used to
antibiotic. Indeed, we show here that replacing the extra- inoculate wells containing TSB with standard twofold incre-
cellular loop of BraE with that of VraE prevented bacitracin ments of antibiotic concentration. Plates were incubated for
10 h with vigorous shaking at 37°C in a Synergy 2 thermoregu-
sensing by the chimeric BraDE*VraE ABC transporter
lated spectrophotometer plate reader using the Gen5TM
(Table 4). The second question is how do BraDE and Microplate Software (BioTek Instruments, Winooski, VT). All
BraSR interact? One possibility is that antibiotic binding experiments were performed in triplicate. The MIC was
and/or transport by BraDE leads to a conformational defined as the concentration at which growth was prevented.
change of the permease structure, promoting interaction
with BraS and subsequent phosphorylation of BraR.
Another unanswered question is how the VraDE ABC DNA manipulations
transporter confers bacitracin resistance. The direction of Oligonucleotides used in this study were synthesized by
bacitracin transport by VraDE remains unknown. This is Sigma-Proligo and their sequences are listed in Table 6.
also the case for the bacitracin transporter BceAB of S. aureus chromosomal DNA was isolated using the
B. subtilis. Some studies favour the hypothesis of an MasterPureTM Gram-positive DNA purification Kit (Epicentre
export system based in particular on the lack of a Biotechnologies). Plasmid DNA was isolated using a QIAprep
substrate-binding protein (Bernard et al., 2007) while Spin Miniprep kit (Qiagen) and PCR fragments were purified
using the Qiaquick PCR purification kit (Qiagen). T4 DNA
others suggest an import function due to the general
ligase and restriction enzymes (New England Biolabs), PCR
architecture of BceAB (Rietkötter et al., 2008). Based on reagents and High-Fidelity Phusion thermostable DNA Poly-
our results and the importance of the large extracytoplas- merase (Roche) were used according to the manufacturer’s
mic domain that appears to be required as a substrate- recommendations. Nucleotide sequencing of plasmid con-
binding protein, we favour the hypothesis that VraDE acts structs was carried out by Genome Express-Cogenics or
as an importer. Bacitracin could be bound by the extra- MilleGen.
cellular loop of the permease and then transferred to the
cytoplasm, away from its site of action. Further work will
Plasmid and mutant construction
be required to unravel the mechanisms involved in detec-
tion and activation of the BraSR/BraDE/VraDE resistance Plasmid pSA14, a derivative of the shuttle vector pMK4 (Sul-
module to confirm these hypotheses. livan et al., 1984), which carries a promoterless E. coli lacZ
gene, was used to construct transcriptional lacZ reporter
fusions (Joanne et al., 2009). Plasmid pMK4-Pprot, a deriva-
Experimental procedures tive of vector pMK4 carrying a constitutively expressed Gram-
positive promoter sequence (Archambaud et al., 2005), was
Bacterial strains and growth procedures used for gene complementation experiments. The thermo-
sensitive shuttle vector pMAD was used for introducing mark-
Escherichia coli K12 strain DH5a™ [F- (f80dlacZDM15)
erless gene deletions (Arnaud et al., 2004). Mutant strains of
D(lacZYA-argF) U169 recA1 endA1 hsdR17 (rk-, mk+) phoA
S. aureus HG001 used in this study were obtained by gene
supE44 l- thi-1 gyrA96 relA1] (Invitrogen) was used for
deletions, removing the entire coding sequence without the
cloning experiments. S. aureus strain HG001, a rsbU + deriva-
introduction of an antibiotic resistance gene. In a first step, a
tive of strain NCTC 8325 (Herbert et al., 2010) and its deriva-
recombination cassette, consisting of two fused DNA frag-
tives were used in all experiments. S. aureus strains and
ments, of approximately 500 bp, corresponding to the chro-
plasmids used in this study are listed in Table 5. Plasmids
mosomal DNA regions located directly upstream and
were constructed in E. coli and passaged through the restric-
downstream from the gene of interest was generated by PCR
tion modification deficient S. aureus strain RN4220 (Kre-
using the gene splicing by strand overlap extension PCR
iswirth et al., 1983) before transformation into strain HG001
technique (SOE-PCR) (Horton et al., 1989) as previously
and its derivatives. E. coli strains were grown in LB medium
described for several of the S. aureus TCS genes (Toledo-
and ampicillin (100 mg ml-1) was added when required.
Arana et al., 2005). Oligonucleotides used for PCR amplifi-
S. aureus was grown in Trypticase Soy Broth (TSB; Difco)
cations are listed in Table 6. Amplicons were cloned into
with shaking (200 r.p.m.) at 37°C, with chloramphenicol
the temperature-sensitive shuttle vector pMAD between the
(10 mg ml-1) or erythromycin (2 mg ml-1) added for plasmid
BamHI/NcoI, BamHI/PstI or BamHI/EcoRI restriction sites.
selection when required. E. coli and S. aureus strains were
For the DvraFG mutant, a 1021 bp DNA fragment corre-
transformed by electroporation using standard protocols
sponding to the Tn554 spc spectinomycin resistance gene
(Sambrook et al., 1989) and transformants were selected on
(Murphy, 1985) was cloned between the upstream and down-
LB or Trypticase Soy Agar (TSA; Difco) plates, respectively,
stream amplicons, yielding pMADvraFG (see Tables 5 and
with the appropriate antibiotics.
6). Nucleotide sequences of the constructs were confirmed
by DNA sequencing. Following introduction into S. aureus
MIC determinations HG001, integration and excision of pMAD derivatives with
deletion of the chromosomal region of interest were carried
Minimal inhibitory concentration determinations were per- out as previously described (Arnaud et al., 2004). Gene dele-
formed in a 96-well microtitre plate (100 ml culture volume). A tions in mutant strains were verified by PCR.

Table 5. Strains and plasmids used in this study.
Strain or plasmid Relevant genotype or description Sourcea
Strains
HG001 NCTC 8325 rsbU + Herbert et al. (2010)
ST1036 DgraRS Joanne et al. (2009)
ST1068 DvraFG::spc, Spr pMADvraFG→HG001
ST1087 DbraRS pSA14-PbraDE–lacZ, Cmr pSA14-PbraDE–lacZ→ST1179
ST1088 DbraRS pSA14-PvraDE–lacZ, Cmr pSA14-PvraDE–lacZ→ST1179
ST1090 DvraDE pMADvraDE→HG001
ST1097 DbraDE pMADbraDE→HG001
ST1163 DvraSR pMADvraSR→HG001
ST1179 DbraRS pMADbraRS→HG001
ST1180 DbraD pMADbraD→HG001
ST1181 DvraD pMADvraD→HG001
ST1182 DbraDE DvraDE pMADbraDE→ST1090
ST1183 DSA2419 pMADSA2419→HG001
ST1184 DbraRS pMK4-Pprot braRS, Cmr pMK4-Pprot braRS→ST1179
ST1121 DbraRS pMK4-Pprot braR, Cmr pMK4-Pprot braR→ST1179
ST1185 DbraRS pMK4-Pprot braDE, Cmr pMK4-Pprot braDE→ST1179
ST1186 DbraRS pMK4-Pprot vraDE, Cmr pMK4-Pprot vraDE→ST1179
ST1187 DbraDE pMK4-Pprot braDE, Cmr pMK4-Pprot braDE→ST1097
ST1188 DbraDE pMK4-Pprot vraDE, Cmr pMK4-Pprot vraDE→ST1097
ST1189 DbraDE pSA14-PbraDE–lacZ, Cmr pSA14-PbraDE–lacZ→ST1097
ST1190 DbraDE pSA14-PvraDE–lacZ, Cmr pSA14-PvraDE–lacZ→ST1097
ST1235 DbraDE pMK4-Pprot braDE*vraE, Cmr pMK4-Pprot braDE*vraE→ST1097
ST1191 DvraDE pMK4-Pprot vraDE, Cmr pMK4-Pprot vraDE→ST1090
ST1192 DvraDE pMK4-Pprot braDE, Cmr pMK4-Pprot braDE→ST1090
ST1193 DvraDE pMK4-Pprot vraFG, Cmr pMK4-Pprot vraFG→ST1090
ST1194 DvraDE pMK4-Pprot vraFG*vraE, Cmr pMK4-Pprot vraFG*vraE→ST1090
ST1234 DvraDE pMK4-Pprot braDE*vraE, Cmr pMK4-Pprot braDE*vraE→ST1090
ST1195 DvraDE pSA14-PbraDE–lacZ, Cmr pSA14-PbraDE–lacZ→ST1090
ST1196 DvraDE pSA14-PvraDE–lacZ, Cmr pSA14-PvraDE–lacZ→ST1090
ST1197 DbraD pMK4-Pprot braD, Cmr pMK4-Pprot braD→ST1180
ST1231 DvraD pMK4-Pprot, Cmr pMK4-Pprot→ST1181
ST1198 DvraD pMK4-Pprot vraD, Cmr pMK4-Pprot vraD→ST1181
ST1205 DvraD pMK4-Pprot vraD*WB, Cmr pMK4-Pprot vraD*WB→ST1181
ST1210 DbraD pMK4-Pprot, Cmr pMK4-Pprot→ST1180
ST1211 DbraD pMK4-Pprot vraD, Cmr pMK4-Pprot vraD→ST1180
ST1206 DbraD pMK4-Pprot braD*WB, Cmr pMK4-Pprot braD*WB→ ST1180
ST1230 DbraD pSA14-PvraDE–lacZ, Cmr pSA14-PvraDE–lacZ→ ST1180
ST1217 DvraDE pMK4-Pprot, Cmr pMK4-Pprot→ST1090
ST1233 DvraDE pMK4-Pprot braR, Cmr pMK4-Pprot braR→ST1090
ST1216 DbraDE pMK4-Pprot, Cmr pMK4-Pprot→ST1097
ST1232 DbraDE pMK4-Pprot braR, Cmr pMK4-Pprot braR→ST1097
ST1240 DbraDE DvraDE pMK4-Pprot braDE, Cmr pMK4-Pprot braDE→ST1182
ST1218 DbraDE DvraDE pMK4-Pprot vraDE, Cmr pMK4-Pprot vraDE→ST1182
ST1238 DbraRS pMK4-Pprot, Cmr pMK4-Pprot→ST1179
ST1239 DbraDE DvraDE pMK4-Pprot, Cmr pMK4-Pprot→ST1182
ST1244 pSA14-PvraSR–lacZ, Cmr pSA14-PvraSR–lacZ→HG001
ST1245 pSA14-PbraDE–lacZ, Cmr pSA14-PbraDE–lacZ→HG001
ST1246 pSA14-PbraRS–lacZ, Cmr pSA14-PbraRS–lacZ→HG001
ST1247 DbraRS pSA14-PbraRS–lacZ, Cmr pSA14-PbraRS–lacZ→ST1179
ST1208 pSA14-PvraDE–lacZ, Cmr pSA14-PvraDE–lacZ→HG001
ST1221 pSA14-PvraDE–lacZDB, Cmr pSA14-PvraDE–lacZDB→HG001
ST1222 pSA14-PvraDE–lacZDC, Cmr pSA14-PvraDE–lacZDC→HG001
ST1223 pSA14-PvraDE–lacZDD, Cmr pSA14-PvraDE–lacZDD→HG001
ST1224 pSA14-PvraDE–lacZDE, Cmr pSA14-PvraDE–lacZDE→HG001
ST1241 pSA14-PbraDE–lacZDB, Cmr pSA14-PbraDE–lacZDB→HG001
ST1242 pSA14-PbraDE–lacZDC, Cmr pSA14-PbraDE–lacZDC→HG001
ST1243 pSA14-PbraDE–lacZDD, Cmr pSA14-PbraDE–lacZDD→HG001
ST1092 DvraSR pSA14-PbraDE–lacZ, Cmr pSA14-PbraDE–lacZ→ST1163
ST1091 DgraRS pSA14-PbraDE–lacZ, Cmr pSA14-PbraDE–lacZ→ST1036
ST1086 DvraSR pSA14-PvraDE–lacZ, Cmr pSA14-PvraDE–lacZ→ST1163
ST1089 DgraRS pSA14-PvraDE–lacZ, Cmr pSA14-PvraDE–lacZ→ST1036
ST1120 HG001 pMK4-Pprot, Cmr pMK4-Pprot→HG001
ST1150 DvraFG::spc pMK4-Pprot, Spr, Cmr pMK4-Pprot→ST1068
ST1151 DvraFG::spc pMK4-PprotvraFG, Spr, Cmr pMK4-PprotvraFG→ST1068
ST1177 DvraFG::spc pMK4-Pprot vraFG*vraE, Spr, Cmr pMK4-Pprot vraFG*vraE→ST1068

Table 5. cont.
Strain or plasmid Relevant genotype or description Sourcea
Plasmids
pSA14 pMK4 derivative carrying promoterless E. coli lacZ for constructing Joanne et al. (2009)
transcriptional fusions, Cmr
pMK4-Pprot pMK4 derivative carrying a constitutive Gram-positive promoter for gene Archambaud et al. (2005)
complementation, Cmr
pMAD pE194 derivative with a thermosensitive origin of replication for Arnaud et al. (2004)
deletion/replacement of genes in Gram-positive bacteria, Err
pMAD braRS pMAD derivative allowing deletion of the braRS genes, Err This study
pMAD braDE pMAD derivative allowing deletion of the braDE genes, Err This study
pMAD braD pMAD derivative allowing deletion of the braD gene, Err This study
pMAD graRS pMAD derivative allowing deletion of the graRS genes, Err This study
pMAD vraFG pMAD derivative allowing deletion of the vraFG genes, Err, Spr This study
pMAD vraSR pMAD derivative allowing deletion of the vraSR genes, Err Toledo-Arana et al. (2005)
pMAD vraDE pMAD derivative allowing deletion of the vraDE genes, Err This study
pMAD vraD pMAD derivative allowing deletion of the vraD gene, Err This study
pMAD SA2419 pMAD derivative allowing deletion of the SA2419 gene, Err This study
pSA14-PbraRS–lacZ pSA14 derivative carrying the promoter region of braRS, Cmr This study
pSA14-PvraSR–lacZ pSA14 derivative carrying the promoter region of vraSR, Cmr This study
pSA14-PbraDE–lacZ pSA14 derivative carrying the promoter region of braDE, Cmr This study
pSA14-PbraDE–lacZDA pSA14 derivative carrying a truncated promoter region of braDE, Cmr This study
pSA14-PbraDE–lacZDB pSA14 derivative carrying a truncated promoter region of braDE, Cmr This study
pSA14-PbraDE–lacZDC pSA14 derivative carrying a truncated promoter region of braDE, Cmr This study
pSA14-PvraDE–lacZ pSA14 derivative carrying a truncated promoter region of vraDE, Cmr This study
pSA14-PvraDE–lacZDA pSA14 derivative carrying a truncated promoter region of vraDE, Cmr This study
pSA14-PvraDE–lacZDB pSA14 derivative carrying a truncated promoter region of vraDE, Cmr This study
pSA14-PvraDE–lacZDC pSA14 derivative carrying a truncated promoter region of vraDE, Cmr This study
pMK4-Pprot braRS pMK4-Pprot derivative carrying the braRS genes, Cmr This study
pMK4-Pprot braDE pMK4-Pprot derivative carrying the braDE genes, Cmr This study
pMK4-Pprot braD pMK4-Pprot derivative carrying the braD gene, Cmr This study
pMK4-Pprot vraDE pMK4-Pprot derivative carrying the vraDE genes, Cmr This study
pMK4-Pprot vraD pMK4-Pprot derivative carrying the vraD gene, Cmr This study
pMK4-Pprot vraFG pMK4-Pprot derivative carrying the vraFG genes, Cmr This study
pMK4-Pprot vraFG*vraE pMK4-Pprot derivative carrying the vraFG*vraE genes, where the extracellular This study
loop-coding sequence of vraG was replaced by that of vraE, Cmr
pMK4-Pprot braDE*vraE pMK4-Pprot derivative carrying the braDE*vraE genes, where the extracellular This study
loop-coding sequence of braE was replaced by that of vraE, Cmr
pMK4-Pprot braD*WB pMK4-Pprot derivative carrying the braD gene containing a single-point This study
mutation in the Walker B motif, Cmr
pMK4-Pprot vraD*WB pMK4-Pprot derivative carrying the vraD gene containing a single-point This study
mutation in the Walker B motif, Cmr
a. Arrows indicate plasmid introduction by electroporation.
For complementation experiments, DNA fragments corre- sites (MF150 and OAH137; upstream fragment) and BsaI/
sponding to the gene-coding sequences were amplified with SalI restriction sites (OAH140 and OAH141; downstream
oligonucleotides generating either BamHI/PstI or BamHI/SalI fragment; see Table 6). Following digestion with BsaI, the two
restriction sites at the extremities as listed in Table 6 and vraG fragments were then ligated to the vraE extracellular
cloned in the pMK4-Pprot replicative plasmid (Archambaud loop sequence, seamlessly fusing the three fragments
et al., 2005). To construct the chimeric system VraG MSD together without adding any additional nucleotides. The
protein with the extracellular loop of VraE (VraG*vraE), the resulting fragment was re-amplified by PCR using the exter-
predicted membrane topologies of VraG and VraE were nal oligonucleotides (MF150 and OAH141), and cloned
determined using the TopPredII program and von Heijne’s between the BamHI and SalI restriction sites of plasmid
algorithm (Claros and von Heijne, 1994). The coding pMK4-Pprot, yielding plasmid pMK4-Pprot vraFG*vraE. A
sequence of the vraE extracytoplasmic loop was amplified by similar strategy was used to generate plasmid pMK4-Pprot
PCR using oligonucleotides OAH138 and OAH139, carrying braDE*vraE, where the coding sequence of the BraE permease
BsaI restriction sites (see Table 6). BsaI is a type IIS restric- extracellular loop was replaced with that of VraE. The
tion endonuclease, which cleaves after its restriction site, sequence of the vraE extracytoplasmic loop was amplified by
generating DNA fragments whose tetranucleotide cohesive PCR using oligonucleotides OAH138 and OAH214 carrying
ends can be defined and designed to be specifically BsaI restriction sites (see Table 6) and DNA fragments cor-
compatible. Two PCR fragments corresponding to the DNA responding to the braE upstream and downstream regions
regions flanking the vraG extracellular loop were generated, were amplified using oligonucleotide pairs OAH105/OAH213
using oligonucleotides containing BamHI/BsaI restriction and OAH215/OAH106 respectively. Single-point mutations in

Table 6. Oligonucleotides used in this study.a
Name Description Sequence
TCS17A braRS upstream region, deletion mutant 5′-GGATCCGCTGCAGAATCAGTAATATT-3′

TCS17B 5′-AAGCTTGATATTAGAAGAAGGAAGTTA-3′
TCS17C braRS downstream region, deletion mutant 5′-AAGCTTAAACTTTCAATATTGTAAGTATA-3′
TCS17D 5′-GAATTCCACGGTATTGTTAGAATTGA-3′
OAH071 braDE upstream region, deletion mutant 5′-CGCGCGGGATCCCTATAAGCCAACAACAACAAATTG -3′
OAH072 5′-AACATGGTTAATTGCGGCAACATGGCATGCACCTC-3′
OAH073 braDE downstream region, deletion mutant 5′-TGCATGCCATGTTGCCGCAATTAACCATGTTTCAGTG-3′
OAH074 5′-CCCGGGCCATGGCGAACATTCTATATAGAGTTTC-3′
OMD189 vraFG upstream region, deletion mutant 5′-GGAGGATCCTCGATGCTGGATACACAGCTG-3′
OMD190 5′-CTCCTCGAGCCAGGCTTTGGCACTTG-3′
OMD191 vraFG downstream region, deletion mutant 5′-AGAAGATCTGCTGTTTTTGCAGTGACGGC-3′
OMD192 5′-GGGGATCCCGAATTCAATAGCAGCACG-3′
OAH075 vraDE upstream region, deletion mutant 5′-GGGCCCGGATCCGACACCTACGAATACAAATGC-3′
OAH076 5′-GTAAGTGTTAAATGGATATCGTCATAGTCTCACTCC-3′
OAH077 vraDE downstream region, deletion mutant 5′-AGACTATGACGATATCCATTTAACACTTACGATTAAAAG-3′
OAH078 5′-CCCGGGCCATGGCTTCCAATGGTGTTCTATAACC-3′
OAH092 braRS promoter region, lacZ fusion 5′-AACATACTGCAGCCTCCTGAGTATATGACTATGTC-3′
OAH102 5′-AGTATCGGATCCCCACCTCAAATTATATTTAATTC-3′
OAH094 vraSR promoter region, lacZ fusion 5′-ACAAAGCTGCAGGAAGAATAGTTCAACATATACTAAG-3′
OAH095 5′-CATAGTGGATCCCTATCACCTTTTATAATAAG-3′
OAH079 braDE promoter region lacZ fusions 5′-CCCGTGCTGCAGGGCCAGCGCCAAAGTAACTC-3′
OAH080 5′-GTGTCTGGATCCGCACCTCATTTCTTTGTTAGTTC-3′
OAH108 DB 5′-CCAGATCTGCAGAAACTTTCAATATTGTAAGTATACTAG-3′
OAH135 DC 5′-AAAAACCTGCAGTATTGTAAGTATACTAGTAAC-3′
OAH136 DD 5′-CAATATCTGCAGTATACTAGTAACATTTTTTTAC-3′
OAH209 DE 5′-CCAGATCTGCAGAAACCGTAAATATTGTAAGTATACTAG-3′
OAH081 vraDE promoter region lacZ fusions 5′-CCCGCGCTGCAGCCATTTAATACCTTGATTTCC-3′
OAH082 5′-GTGCCCGGATCCCTCACTCCTTTTGTATTTAATTTC-3′
OAH109 DB 5′-AAAATCCTGCAGGTAGCGATAGCTTTCAACTCTGTAAGG-3′
OAH122 DC 5′-GATAGCCTGCAGCTCTGTAAGGTTCATTCATTG-3′
OAH121 DD 5′-CAACTCCTGCAGGTTCATTCATTGAATTGTAAG-3′
OAH210 DE 5′-AAAATCCTGCAGGTAGCGATAGCCGTAAACTCTTTCGGG-3′
OAH100 braRS-coding sequence, complementation 5′-GATAATGGATCCGAAGGAAGAAGATTATAGATG-3′
OAH110 5′-ACAATAGTCGACGTTTTTATTCATCTGGAAATTG-3′
OAH105 braDE-coding sequence, complementation 5′-AGAACTGGATCCGAAATGAGGTGCATGCCATGTTGC-3′
OAH106 5′-ATTTCACTGCAGCATGGTTAATTGCGTTGTTGATG-3′
OAH103 vraDE-coding sequence, complementation 5′-TGAAATGGATCCCAAAAGGAGTGAGACTATGACG-3′
OAH104 5′-GCATCTCTGCAGCGTAAGTGTTAAATGGTTTTC-3′
MF150 vraFG-coding sequence, complementation 5′-GGAGGATCCGATAAATTATAGGAGTGTTAAAGTG-3′
MF151 5′-CTGCTGCAGTTATATGGAATGTCTAATTGT-3′
OAH137 vraFG*vraE-coding sequence, complementation 5′-AGGGGTCTCCATTTACTTATAGCAGCAAAGCAAAGAAC-3′
OAH138 5′-GGCGGTCTCCAAATCAAATACAGATCAAACCCTTAC-3′
OAH139 5′-AATGGTCTCCCGGTGTTAGTAGCATCGACCTC-3′
OAH140 5′-AATGGTCTCCACCGGAATATTATTATTTGTAAC-3′
OAH141 5′-CTGGTCGACTTATATGGAATGTCTAATTGT-3′
OAH173 5′-ACAATCCTGCAGTCATATATTGACACTTCCTCC-3′
OAH197 braD Walker B motif-coding sequence site-directed mutagenesis 5′-GTTTTTGAATCAAGTGCACCTGTAGGTTGATCGGCTAG-3′
OAH198 5′-TCAACCTACAGGTGCACTTGATTCAAAAACATCAAAGGC-3′
OAH196 5′-AAAACGGTCGACTTAAATGTCATTTGAGACACC-3′
OAH199 vraD Walker B motif-coding sequence site-directed mutagenesis 5′-TGCACTTTTCGAGTCGAGTGCGCCTGTTGGTTGATCTGC-3′
OAH200 5′-CCAACAGGCGCACTCGACTCGAAAAGTGCAAATGACCTA-3′
OAH213 braDE*vraE-coding sequence, complementation 5′-TCTGGTCTCCATTTGGTTGTTAATGTCATTGTGAGCG-3′
OAH214 5′-AATGGTCTCCATATGTTAGTAGCATCGACCTC-3′
OAH215 5′-AATGGTCTCCATATTTGTAGGTGGCGTAGTATC-3′
OSA161 16S rRNA intragenic region, qRT-PCR 5′-ACGTGGATAACCTACCTATAAGACTGGGAT-3′
OSA162 5′-TACCTTACCAACTAGCTAATGCAGCG-3′
OAH123 braDE intragenic region, qRT-PCR 5′-CGGCGGATACTTACTTGG-3′
OAH124 5′-CTACAGAGTCAAATGGATAATACC-3′
OAH125 vraDE intragenic region, qRT-PCR 5′-GCACTAACTATGGCTATGACATC-3′
OAH126 5′-TGAATACAACATCGGTAATAGATACG-3′
OAH129 braRS promoter region, primer extension 5′-AACCATCCTGTTTCAGTCGTATC-3′
OAH134 5′-GTGCTGTCGTAGCATTCAAACCTTTACCATAAACC-3′
OAH127 vraDE promoter region, primer extension 5′-CCCTTTTCTCAATTTACACAAAC-3′
OAH142 5′-CGCAACGAATTCGCCTTTTTGTATGTCAAAG-3′
OMD156 Tn554 spc spectinomycin resistance gene amplification, gene deletion 5′-AGAAGATCTCACCTAGATCCTTTTGACTC-3′
OMD157 5′-CTCCTCGAGAAAGTAAGCACCTGTTATTGC-3′
a. Added restriction site sequences are shown in italics.

the braD and vraD Walker B motif-coding sequences were Primer extensions
introduced by site-directed mutagenesis through SOE-PCR
(Ho et al., 1989). Briefly, two DNA fragments were generated Primer extensions were performed as previously described
by PCR. The first contained the upstream region and the (Chastanet et al., 2001) using 40 mg of RNA, 2 pmol of oligo-
single-point mutation obtained using a reverse oligonucle- nucleotide (previously radiolabelled with [g-32P]-ATP using T4
otide containing 1 mismatch (Table 6, OAH105/OAH197 and polynucleotide kinase, New England Biolabs) and 200 U of
OAH103/OAH199 for braD and vraD respectively). The Superscript II reverse transcriptase (Invitrogen). Oligonucle-
second fragment, corresponding to the region downstream of otides were chosen so as to hybridize approximately
the mutation site (Table 6, oligonucleotides OAH198/ 30–70 bp downstream from the translation initiation codon
OAH173 and OAH200/OAH196 for braD and vraD respec- (see Table 6). The corresponding DNA sequencing reactions
tively), had a 30 bp overlap with the first one, allowing the two were carried out with the same oligonucleotides and PCR-
to be joined by gene splicing through SOE-PCR, and the amplified DNA fragments carrying the respective promoter
resulting DNA fragment was cloned in plasmid pMK4-Pprot, regions, using the Sequenase PCR product sequencing kit
yielding plasmids pMK4-Pprot braD*WB and pMK4-Pprot (USB, Cleveland, OH).
vraD*WB.
For constructing transcriptional lacZ fusions, promoter
regions of the braDE, SA2419-braRS, vraDE and vraSR cDNA synthesis and qRT-PCRs
operons were amplified by PCR using oligonucleotides intro-
cDNAs were obtained using the iScript cDNA synthesis kit
ducing BamHI/PstI restriction sites (see Table 6). The corre-
(Bio-Rad, Hercules, CA) according to the manufacturer’s rec-
sponding DNA fragments were then cloned between the
ommendations, in a final reaction volume of 20 ml containing
corresponding restriction sites of the pSA14 vector, yielding
1 mg of total RNA. The mixture was incubated for 5 min at
plasmids PbraDE–lacZ (PSA2415-2416–lacZ), PSA2419-braRS–lacZ
25°C, 30 min at 42°C and reverse transcriptase was then
(PSA2417-2419–lacZ), PvraDE–lacZ and PvraSR–lacZ. To construct
inactivated by heating the mixture at 85°C for 5 min. For
the promoter regions with the three point mutations in the
qRT-PCR experiments, primers were designed using
BraR operator site, PbraDE–lacZDE and PvraDE–lacZDE, the cor-
BEACON Designer 4.02 software (Premier Biosoft Interna-
responding DNA fragments were amplified by PCR using
tional, Palo Alto, CA) to amplify 200–300 bp amplicons (see
forward oligonucleotides containing three mismatches (oligo-
Table 6). qRT-PCRs, critical threshold cycles (CT) and n-fold
nucleotides OAH209 and OAH210, for braDE and vraDE
changes in transcript levels were performed and determined
respectively; Table 6).
as previously described and normalized with respect to 16S
rRNA whose levels did not vary under our experimental con-
ditions (Dubrac et al., 2007).
b-Galactosidase assays
For b-galactosidase assays, cells were grown in TSB until

OD600 = 2. Bacitracin was added to the medium at different
Acknowledgements
sublethal concentrations when required. S. aureus carrying This work was supported by research funds from the
lacZ fusions were harvested by centrifuging 2 ml of culture European Commission [StaphDynamics (LHSM-CT-2006-
samples (2 min; 20 800 g). Cells were resuspended in 500 ml 019064) and BaSysBio (LSHG-CT-2006-037469) grants], the
of Z buffer (Miller, 1972) with 0.5 mg ml-1 DNase and Centre National de la Recherche Scientifique (CNRS URA
0.1 mg ml-1 lysostaphin added extemporaneously, and lysed 2172), Agence Nationale de la Recherche (ANR GrabIron
by incubation at 37°C for 30 min. Cell debris were eliminated and NaBab) and the Institut Pasteur (PTR N°256 and PTR
by centrifugation (2 min; 20 800 g) and the supernatant N°336). We are grateful to Cécile Wandersman for critical
was either used directly for assays or stored at -20°C. reading of the manuscript. We thank Olivier Poupel for assis-
Assays were performed as previously described and tance with qRT-PCR experiments and Iñigo Lasa Uzcudun
b-galactosidase-specific activities were expressed as Miller (Universidad Pública de Navarra-CSIC, Pamplona, Spain) in
units per mg protein (Miller, 1972). Protein concentrations whose laboratory part of this work was carried out. Mélanie
were determined using the Bio-Rad protein assay (Bio-Rad, Falord received a Young Scientist Fellowship from the
Hercules, CA) (Bradford, 1976). All experiments were carried Conseil Pasteur-Weizmann.
out in triplicate.
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Molecular Microbiology - 2011 - Hiron - Bacitracin and Nisin Resistance in Staphylococcus Aureus A Novel Pathway Involving

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Molecular Microbiology (2011) 81(3), 602–622 䊏 doi:10.1111/j.1365-2958.2011.07735.

Bacitracin and nisin resistance in Staphylococcus aureus:

© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 81, 602–622

© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 81, 602–622

© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 81, 602–622

© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 81, 602–622

HG001 NCTC 8325 rsbU+ 48 > 128

© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 81, 602–622

Fig. 4. A highly conserved imperfect

© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 81, 602–622

© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 81, 602–622

Strain Relevant genotype MIC bacitracin (mg ml-1)

ST1210 DbraD pMK4-Pprot 6

As shown above, VraDE plays a central role in bacitra-

© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 81, 602–622

© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 81, 602–622

Fig. 7. Signal transduction network

© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 81, 602–622

© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 81, 602–622

© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 81, 602–622

© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 81, 602–622

Table 5. Strains and plasmids used in this study.

Strain or plasmid Relevant genotype or description Sourcea

© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 81, 602–622

Strain or plasmid Relevant genotype or description Sourcea

a. Arrows indicate plasmid introduction by electroporation.

© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 81, 602–622

Table 6. Oligonucleotides used in this study.a

Name Description Sequence

TCS17A braRS upstream region, deletion mutant 5′-GGATCCGCTGCAGAATCAGTAATATT-3′

a. Added restriction site sequences are shown in italics.

© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 81, 602–622

For b-galactosidase assays, cells were grown in TSB until

© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 81, 602–622

© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 81, 602–622

© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 81, 602–622

© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 81, 602–622

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